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Abstracts / Journal of Biotec

different pH buffers. When time of reaction is longer, the conversionof �-cyclodextrin–poly(ethylene glycol) is higher. Crystals obtainedby this method were observed by scanning electron microscopyand X-ray diffraction and FTIR. SEM images show that the shapeof crystals at 0 ◦C are rod-like; by increasing the temperatureto 25 ◦C their shape became random. If temperature rises up to40 ◦C temperature effect changes the random condition which canattribute special shape to them. On the other hand by increas-ing the temperature their hexagonal or cubic shape changed. ButpH has no specific effect on the crystal shape and growth, onlycan express by increasing pH from 7 to 9 the size of crystalsbeen smaller. By increasing the time of reaction crystals thicknessincreased.

Fig. 1 Effect of temperature on the shape and size of crystals atpH 7 and time of reaction is 2 h.

Fig. 2 Effect of time of reaction on the shape and size of crystalsat pH 7.

Fig. 3 Effect of pH on the shape and size of crystals (time ofreaction: 6 h at 25 C): (a) pH 7 and (b) pH 9.

Keywords: �-Cyclodextrin; Poly(ethylene glycol) nanocrystalinclusion complex; Crystal growth

doi:10.1016/j.jbiotec.2008.07.1457

VII2-P-030

The expression of formate dehydrogenases and hydrogenases inSyntrophobacter fumaroxidans in different growth phases andat different pHs

Xu Cheng ∗, Petra Worm, Alfons J.M. Stams, Caroline M. Plugge

Wageningen University, Laboratory of Microbiology, Dreijenplein 10,6703 HB Wageningen, The Netherlands

E-mail address: xu.cheng@wur.nl (X. Cheng).

Under anaerobic conditions, the complex organic matter degra-dation process cannot be completed by one bacterium. Instead,specialized syntrophic microorganisms play their own specificrole. Propionate is an important intermediate in anaero-bic degradation and is used as substrate by Syntrophobacterfumaroxidans.

S. fumaroxidans is a non-motile, non-spore-forming, Gram-negative propionate degrading �-proteobacterium, which wasisolated from a culture enriched from anaerobic granular sludge.

It can oxidize propionate syntrophically in co-culture with thehydrogen- and formate-utilizing Methanospirillum hungateii andMethanobacterium formicicum, but it is also able to oxidize propi-onate and a few other organic compounds in pure culture withsulfate or fumarate as the electron acceptor (Stams and Dong,1995). S. fumaroxidans’ genome, sequenced by DOE Joint GenomeInstitute, contains four formate dehydrogenases (FDH) codinggene clusters. FDH-1, FDH-2 and FDH-4 each contain a SeCysresidue in the amino acid sequence whereas FDH-3 contains aconventional Cystein. S. fumaroxidans’ genome also encodes sixputative hydrogenase (H2ase) gene clusters: three [NiFe]-H2ases,two [NiFeSe]-H2ases and one [FeFe]-H2ase. The differences inexpression of [NiFe] and [NiFeSe] H2ase may be regulated bypH.

The gene expression level in different growth phases alsomay be different; normally it was measured in late log phase,early and mid stages were followed in this research. S. fumaroxi-dans was cultivated in pure cultures with hydrogen–fumarate andformate–fumarate at various pHs (6.0–8.0). Growth curves weremade according to the OD, GC and HPLC measurements. However,gene expression experiments to measure pH influence are still in

gy 136S (2008) S620–S632 S629

process. Furthermore cells were harvested in early, mid and endlog phases, and gene expression levels were measured. Prelimi-nary results suggest that the expression of FDH-1 is 10 times lowerin hydrogen-fumarate grown cells compared to formate–fumarategrown cells. Hydrogenase expression, however, seems similar inboth growth substrates.

Reference

Stams, A.J.M., Dong, X., 1995. Role of formate and hydrogen in the degradation ofpropionate and butyrate by defined suspended cocultures of acetogenic andmethanogenic bacteria. Antonie van Leeuwenhoek 68, 281–284.

doi:10.1016/j.jbiotec.2008.07.1458

VII2-P-031

Morphological–molecular identification and evaluation ofmicrocystin synthetase gene E (mcyE) of cyanobacteria in Kras-noyarsk reservoir

Igor Kozhevnikov ∗, Nelya Kozhevnikova, Nataliya Ivanova

Siberian Federal University, Krasnoyarsk, Russia

E-mail address: ikozhevnikov@inbox.ru (I. Kozhevnikov).

To date, planktonic cyanobacteria of large Siberian water ecosystem– the Krasnoyarsk reservoir – have only been studied by tradi-tional microscopic techniques and not by molecular approaches.The aim of this study was to establish pure cultures of some plank-tonic cyanobacteria from Krasnoyarsk reservoir, to identify strainson the basis of a molecular and morphological study and to teststrains and environmental samples for the detection of potentialmicrocystin producers. The methods of hydrobiology, microbiologyand molecular biology are combined in this work. DNA manipula-tion were done by standard methods and by applying commercialkits and reagents. The similarities between 16S rRNA, mcyE genesin GenBank and the PCR cloning products were analyzed withNCBI sequence similarity search tool (BLAST) and aligned to knownsequence matches using ClustalX. During this study, a total of20 cyanobacterial axenic strains from Krasnoyarsk reservoir wereisolated including genera Anabaena, Aphanizomenon, Oscillato-ria, Planktothrix, Pseudanabaena, Synechococcus, Aphanothece, andMerismopedia. These strains were identified based on the tra-ditional classification system (Komárek and Anagnostidis, 1999;

Anagnostidis and Komárek, 1988) and the genotypic study as partial16S rRNA gene sequences. Four isolates were assigned to com-mon bloom-forming genera. Strains from the genera Anabaena,Oscillatoria, Planktothrix, and Aphanothece, as well as environmen-tal samples from different sites of resorvoir were screened withgeneral and genus-specific microcystin synthetase E gene (mcyE)PCR (Rentala et al., 2006). Two potential toxic strains Oscillatoriasp. KRNA4-05 and Aphanothece sp. KRNA3-01 have been revealed.In particular, the Anabaena lemmermannii that caused the waterreservoir blooming in 2007 is non-toxic. The results showed theimportance of the molecular approach in order to improve the tax-onomy of cyanobacteria of the Krasnoyarsk reservoir and to detectof potential microcystin producers with the purpose of ecosystemcondition monitoring and water quality evaluation.

References

Anagnostidis, K., Komárek, J., 1988. Modern approach to the classification system ofcyanophytes. 3. Oscillatoriales. Algol. Stud. 50–53, 327–472.

Komárek, J., Anagnostidis, K., 1999. Cyanoprokaryota 1. Teil: Chroococcales. In: Ettl,H., Gärtner, G., Heynig, H., Mollenhauer, D. (Eds.), Süsswasserflora von Mitteleu-ropa band 19/1. Gustav Fischer, Berlin, 548 pp.

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for explaining these findings was provided by the protein local-ization studies, which revealed that the AtGluRS-GUS was strictlylocalized in the cytoplasm, and the ecotopic expressed AtHB6-GUSwas in the nucleus, while the co-expression of both AtGluRS andAtHB6-GUS resulted in both nuclear and cytoplasmic localization ofAtHB6-GUS. Thus, the AtGluRS is postulated to interact with AtHB6in cytoplasm and reduce the nuclear compartmentation of AtHB6,

S630 Abstracts / Journal of Biotec

Rentala, A., Rajaniemi-Wacklin, P., Lyra, H., Lepistö, L., Rentala, J., Mankiewicz-Boczek, J., Sivonen, K., 2006. Detection of microcystin-producing cyanobacteriain Finnish lakes with genus-specific microcystin synthetase gene E (mcyE)PCR and associations with environmental factors. Appl. Environ. Microbiol. 72,6101–6110.

doi:10.1016/j.jbiotec.2008.07.1459

VII2-P-032

Evaluation of thiram induced oxidative stress, immunosuppres-sion and apoptosis in avain lymphocytes culture system

Sonu Ambwani 1,∗, Tanuj Kumar Ambwani 1, R.S. Chauhan 2

1 G.B. Pant University of Agriculture & Technology, Pantnagar, India2 Indian Veterinary Research Institute, Izatnagar, India

E-mail address: sonuambwani@yahoo.co.in (S. Ambwani).

Today chemical pesticides are playing vital role in controlling agri-cultural, industrial, home garden and public health pests globally.At the same time these pesticides are constantly contaminatingenvironment and adversely affecting animal and human health.Several studies have reported immunosuppressive effects of car-bamate group of pesticides in animals. But few such reports areavailable employing in vitro system of avian splenocytes.

Thiram is dithiocarbamate fungicide with good avian and mam-malian repellent properties; and a metabolic poison of low acutetoxicity to mammals and a skin irritant (WHO/FAO, 1985). It isalso used as a promoter of vulcanization in the rubber industry,an activator in plastics manufacturing and as a chemosterilantin plastic film dry wound dressing. Thiram has been reported asreproductive teratogenic, mutagenic and tumerogenic (Gahukar,1999). Present communication evaluates oxidative stress, immuno-suppression and apoptosis in chicken lymphocytes due to in vitroexposure of low level dose of thiram employing nitric oxide esti-mation, lymphocyte proliferation assay, cytokine assay, annexin Vassay and DNA fragmentation assay, respectively.

Chicken lymphocytes displayed elevated level of nitric oxidewhen exposed to 1000 times dilution of low-level dose of thiram.Significant decrease in B lymphocytes, T lymphocytes, interleukinI and II levels was also observed in thiram-treated cells as com-

pared to control cells. Thus thiram was found to be immunotoxiceven at low dose level. Annexin V assay revealed increased num-ber of lymphocytes under going apoptosis. It was further confirmedby agarose gel electrophoresis of genomic DNA of thiram-exposedcells which displayed typical apoptotic DNA ladder pattern. It is alsoworthwhile to mention that such in vitro tests in immunotoxicol-ogy can be useful in the quick preliminary evaluation and toxicityassessment of pesticides and will be helpful in reducing the use oflaboratory animals.

Keywords: Thiram; Immunosuppression; Oxidative stress;Cytokine assay; Apoptosis; Cytokines; Avian lymphocytes

References

Gahukar, R.T., 1999. Agro-medical Guide of Synthetic Pesticides. Agri-HorticulturalPublishing House, Nagpur.

World Health Organization/Food and Agriculture Organization, 1985. Data Sheetson Pesticides No. 71. Thiram. WHO/FAO.

doi:10.1016/j.jbiotec.2008.07.1460

gy 136S (2008) S620–S632

VII2-P-033

AtGluRS functions as a positive regulator in ABA signal pathway

Yulin Tang 1,∗, Erwin Grill 2

1 College of Life Science, Shenzhen University, Shenzhen 518060, China2 Technische Universitaet Muenchen, Freising D-85354, Germany

E-mail address: yltang@szu.edu.cn (Y. Tang).

Abscisic acid (ABA) plays crucial roles in various aspects of plantgrowth and development, as well as in adaptation to adverse envi-ronmental stresses. A series of ABA signal components have beencharacterized. The key component of ABA signaling ABI1 regulatesseveral ABA responses. As a target of the PP2C ABI1, the Ara-bidopsis thaliana homeobox protein AtHB6 functions down-streamof ABI1 and acts as a negative regulator in ABA signal pathway(Himmelbach et al., 2002).

To identify more ABA signaling components, the screening forthe interaction partners of AtHB6 was performed. And the Arabidop-sis thaliana glutamyl-tRNA synthetase (AtGluRS) were identifiedthrough screening of the arabidopsis cDNA libraries in a yeasttwo-hybrid system by using the N-terminal AtHB6 as the bait.Further studies in yeast three-hybrid system and the in vivo co-immunoprecipitation revealed that the AtGluRS interacts not onlywith AtHB6 but also with ABI1. It suggested that the complicatedinteraction presented among AtHB6, AtGluRS and ABI1.

The function of the interaction between AtGluRS, AtHB6 andABI1 was surveyed using maize protoplast transient expressionsystem. The co-expression of ABI1 with AtHB6 elevated two foldthe AtHB6-activated gene expression, while the AtGluRS exertedan inhibition in the AtHB6-activated expression of the reportergene. Results demonstrate a positive regulatory role of ABI1 anda suppression effect of AtGluRS upon the AtHB6 function. A basis

consequently the function of AtHB6 as the transcriptional regula-tor is suppressed. The results unravel a new function of AtGluRS asa positive regulator in ABA signal pathway through inhibiting thefunction of the ABA negative regulator AtHB6.

Keywords: AtGluRS; AtHB6; Abscisic acid; Signal transduction

Reference

Himmelbach, A., Hoffmann, T., Leube, M., Hohener, B., Grill, E., 2002. Homeodomainprotein ATHB6 is a target of the protein phosphatase ABI1 and regulates hormoneresponses in Arabidopsis. EMBO J. 21, 3029–3038.

doi:10.1016/j.jbiotec.2008.07.1461